Fig. 7: Structure-based model for NanoLuc luciferase action.

I. A substrate molecule enters the intra-barrel catalytic site, the luciferase retains an “open β-barrel” conformation. II. Catalytic conversion of the substrate into its reaction product, followed by the emission of a blue photon. III. Release of the product out of the intra-barrel catalytic site. This step is accompanied by an open-to-closed conformational transition and a rebinding of the product to the newly formed allosteric site on the protein surface. IV. Dissociation of the product from the surface allosteric site, allowing recycling into the pre-catalytic “open β-barrel” state. The PDB ID codes for representative crystallographic structures are provided.